Project summary/abstract Glycosylation plays vital roles in many cellular events, including protein folding, pathogen recognition, and cancer metastasis. The structural complexity and diversity of glycans parallel their diverse functions. Whereas the primary structures of linear biopolymers, such as proteins and oligonucleotides, are uniquely defined by their one-dimensional sequence, full structural characterization of a glycan requires determination of its two- dimensional topology, linkage and stereochemical configurations. Further analytical challenges arise from the non-template-driven nature of glycan biosynthesis, resulting in glycomes comprising a repertoire of closely- related structures, many of which structural isomers. Recently, a number of electron activated dissociation (ExD) methods have been developed in mass spectrometry laboratories for glycan analysis. Electron capture dissociation (ECD), electron transfer dissociation (ETD), and electronic excitation dissociation (EED) can yield rich structurally informative fragment ions for glycans analyzed in the positive ionization mode. In the negative ionization mode, electron detachment dissociation (EDD) and negative ETD (NETD) are powerful fragmentation methods for sequencing of acidic glycosaminoglycans. Meanwhile, ion mobility spectrometry (IMS) has been applied to separation of glycans. As a post-ionization, gas-phase separation method, IMS complements solution-phase separation methods such as capillary electrophoresis (CE) and liquid chromatography (LC), and can achieve isomer resolution based on differences in their gas-phase conformations. However, conventional drift-time IMS separation occurs on too short a time-scale to be compatible with the slower ExD analysis methods. A new IMS technique, termed trapped ion mobility spectrometry (TIMS), was recently introduced by Bruker Daltonics. We have demonstrated successful coupling of TIMS to high-performance Fourier-transform ion cyclotron resonance (FTICR) MS instrument for separation and identification of glycan linkage isomers. Here, we propose to modify the TIMS device and its control software, for improved mobility resolution, increased m/z operating range, and better integration with ExD-FTICR MS/MS analysis. We will then utilize the improved TIMS-ExD method for detailed structural characterization of glycans. We will also use TIMS-ExD MS/MS in conjunction with off-line LC fractionation to produce a library that contains identified glycan structures with their collision cross section values. This library will be made available to public. The initial development will be carried out on the FTICR MS platform, as it offers superior mass accuracy and resolving power, as well as the best ExD performance. The technology we develop here can later be transferred to other, more affordable MS instruments, following the development of alternative ECD cells to bring the ExD capability to non-ICR instruments.